PROJECT SUMMARY: Rickettsial diseases caused by pathogens of the Anaplasmataceae family, including members of the genera Ehrlichia and Anaplasma, have become a growing public health concern over the past three decades and are a leading cause of tick-borne infections in humans throughout the USA and many parts of the world. The diseases include the human monocytic and granulocytic ehrlichiosis caused by Ehrlichia chaffeensis and Ehrlichia ewingii respectively, and the human granulocytic anaplasmosis resulting from Anaplasma phagocytophilum infections. E. canis, first described as a canine pathogen, also causes infections in people. Recently, another tick-borne pathogen, Ehrlichia muris subsp. eauclairensis, is also reported as causing disease in people. These pathogens have evolved strategies to evade host immunity and cause persistent infections. People with persistent infections are difficult to diagnose and pose risk to blood and organ recipients. Through our recently established mutagenesis experiments, we created E. chaffeensis mutants that contained insertions causing functional gene disruptions. An insertion mutation in the ECH_0660 gene resulted in the pathogen's rapid clearance from two vertebrate hosts. Vaccination with this attenuated mutant induced a strong host response and offered complete against blood stream infection and tick transmission challenges with wild-type E. cahffeensis. Our further studies suggest that the ECH_0660 homologs are well conserved among different Ehrlichia/Anaplasma species. Together, our extensive research forms the strong foundation and premise for the proposed project. In this proposal, we will test the hypothesis that attenuation through a functional deficiency in the E. chaffeensis ECH_0660 gene and its homologs in other related rickettsials will result in safe and efficacious vaccines that are sufficient to prevent wild type infection into the blood stream or from tick transmission; the two possible means by which tick-borne rickettsials cause infections in people. We propose the following three specific aims: 1) Evaluate the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wild type infection challenge through blood stream and tick-transmission. 2) Evaluate the protection of the MLAV against genetically distinct E. chaffeensis strains. 3) Evaluate ECH_0660 gene homolog mutants in related Ehrlichia and Anaplasma species for their efficacy as live attenuated vaccines in conferring protection against the pathogens' infection into blood stream and by tick-transmission. The goals represent a logical extension of the substantial progress we have made from our ongoing research. Further, we have included a rigorous experimental plan to execute the project goals, which are critical in advancing the development of vaccines to prevent diseases caused by several important tick-transmitted Ehrlichia and Anaplasma species pathogens. We have the high-level expertise and the necessary resources to ensure the successful execution of the proposed project goals.